Characterization of ZmPMP3g function in drought tolerance of maize

The genes enconding proteins containing plasma membrane proteolipid 3 (PMP3) domain are responsive to abiotic stresses, but their functions in maize drought tolerance remain largely unknown. In this study, the transgenic maize lines overexpressing maize ZmPMP3g gene were featured by enhanced drought tolerance; increases in total root length, activities of superoxide dismutase and catalase, and leaf water content; and decreases in leaf water potential, levels of O2−·and H2O2, and malondialdehyde content under drought. Under treatments with foliar spraying with abscisic acid (ABA), drought tolerance of both transgenic line Y7-1 overexpressing ZmPMP3g and wild type Ye478 was enhanced, of which Y7-1 showed an increased endogenous ABA and decreased endogenous gibberellin (GA) 1 (significantly) and GA3 (very slightly but not significantly) and Ye478 had a relatively lower ABA and no changes in GA1 and GA3. ZmPMP3g overexpression in Y7-1 affected the expression of multiple key transcription factor genes in ABA-dependent and -independent drought signaling pathways. These results indicate that ZmPMP3g overexpression plays a role in maize drought tolerance by harmonizing ABA-GA1-GA3 homeostasis/balance, improving root growth, enhancing antioxidant capacity, maintaining membrane lipid integrity, and regulating intracellular osmotic pressure. A working model on ABA-GA-ZmPMP3g was proposed and discussed.

www.nature.com/scientificreports/ ZmRCI2-3, ZmRCI2-8, and ZmRCI2-9. Eight ZmPMP3 proteins are categorized into three groups 11 , of which ZmPMP3-1, ZmPMP3-5, ZmPMP3-7 and ZmPMP3-8 belong to the group I, ZmPMP3-4 and ZmPMP3-6 are in the group II, and ZmPMP3-2 is under the group III 11 . Up to now, ZmPMP3s are documented to function in salt tolerance 11 . The ZmRCI2s are considered to be related to drought tolerance because the expression is responsive to drought 12 . Nevertheless, there is no functional experimental evidence, and the detailed mechanisms of the ZmPMP3s in drought tolerance of maize are still unknown. Based on the transcriptome of maize inbred line YQ7-96 under salt stress, we cloned an early drought stress-inducible gene (GenBank accession no. EC869579.1), numbered RA33G4 and now named ZmPMP3g, which encods a PMP3 domain protein 13 . We inferred that ZmPMP3g could likely confer drought tolerance of maize. To confirm the assumption, in the present study, ZmPMP3g function in drought tolerance of maize was investigated.

Results
ZmPMP3g gene. The ZmPMP3g had 99% sequence identity with a LTI6B (GenBank accession no. EU961419.1/EU954644.1) and 97% sequence identity with an early drought induced protein gene (GenBank accession no. NM_001114162) of maize at nucleotide level, respectively. The encoded protein ZmPMP3g was composed of 58 amino acid residues, showing 100% identity with an early drought-inducible protein (GenBank accession no. ABY71210.1) and ZmPMP3-1 protein (EU364508) of maize at amino acid level, respectively.
The expression of genes related to ABA-dependent and -independent signaling (ADIDS) pathways in Y7-1 and Y478. Transcriptome sequencing analyses were conducted on leaves of transgenic line Y7-1 and wild type Y478 pot-grown under control, both control and exogenous ABA (control-exogenous ABA), moderate drought, and moderate drought-exogenous ABA, respectively. As a result, application of exog-   Table S3). There were 59 ADIDS-related DEGs found in Y7-1 leaves against Y478 under control (Table 1). However, the number of the related DEGs was much lower in Y7-1 under treatments with exogenous ABA than under treatments without exogenous ABA (Table 2). , and LWP (c) of pot-grown maize. Analyses were conducted on the second day after the pot mix moisture was at the upper threshold of 50% for moderate drought and 40% for severe drought, respectively. Transgenic plants analyzed were from T4-generation lines. The leaf wilting index for each line under each treatment was calculated based on all leaves of 5 individual plants selected at random. The RWC and LWP were analzyed by using the second fully-expanded leaves down from the top of 3 individual plants selected at random under moderate drought and presented as the mean ± SD. The single asterisk (*) and double asterisk (**) indicated significant differences at p < 0.05 and p < 0.01, respectively. C7-2, Maize inbred line Chang7-2; C-7-2, C7-2 overexpressing ZmPMP3g; HZ4, Maize inbred line Huangzao4; H2-3, HZ4 overexpressing ZmPMP3g; LWP, Leaf water potential; RWC, Relative water content; SD, Standard deviation; T4, Transgenic generation 4; Y478, Maize inbred line Ye478; Y7-1, Y478 overexpressing ZmPMP3g; Z58, Maize inbred line Z58; Z3-1, Z58 overexpressing ZmPMP3g. The total root length of pot-grown maize. Analyses were conducted on the third day after the pot mix moisture was at the upper threshold of 40% for severe drought. Transgenic plants analyzed were from T4-generation lines. The data of transgenic line Z3-1 and wild type Z58 were not available. Each datum was the mean ± SD from root systems of 5 individual plants selected at random. The single asterisk (*) indicated a significant difference at p < 0.05. C7-2, Maize inbred line Chang7-2; C-7-2, C7-2 overexpressing The expression of genes related to gibberellin (GA) in Y7-1. By results of transcriptome sequencing, DEGs related to GA biosynthesis and catabolism were found (Table 3). Of these genes, the expression of GA2ox7, GA2ox12 and GA20ox5 was up-regulated. The expression of one DELLA protein RGA gene was found to be down-regulated in Y7-1 against Y478 under the moderate drought-exogenous ABA treatment (Table 3). Several DEGs encoding Skp1, CUL1, and F-box proteins, which were related to DELLA degradation, were found in Y7-1 (Table 3).  www.nature.com/scientificreports/ Changes in endogenous GA and ABA levels in Y7-1 and Y478. GA and ABA contents were also analyzed with leaves of transgenic line Y7-1 and wild type Y478 pot-grown under control, control-exogenous ABA, moderate drought, and moderate drought-exogenous ABA, respectively. In order to reflect the change tendency of GA and ABA, the fold changes in ABA and GA levels under treatments with foliar spraying with exogenous ABA in comparison with those in respective maize line under treatments without exogenous ABA were analyzed if the endogenous ABA and GA levels under corresponding treatments without exogenous ABA were set to 1 as control. As a result, the endogenous ABA levels significantly increased, GA1 levels decreased significantly under control-exogenous ABA treatment and slightly but not significantly under moderate drought-exogenous ABA treatment, and GA3 levels decreased very slightly but not significantly, in leaves of Y7-1 (Fig. 7d). However, endogenous ABA levels were relatively lower, and both GA1 and GA3 levels did not significantly change in leaves of Y478 under treatments (Fig. 7d).

Discussion
ZmPMP3g was a homologous gene of ZmPMP3-1, which belongs to the group I in ZmPMP3 family according to amino acid sequence 11 . The enhanced drought tolerance (Fig. 2) and the increased total root length ( Fig. 4) of transgenic maize lines suggest that ZmPMP3g plays a role in both drought tolerance and root growth of maize. The increase in root length is undoubtedly beneficial to the uptake and utilization of water in deep soil by plants under drought, which is usually driven by hydrotropism 15 . Taken together with the function of maize ZmPMP3-1 in salt tolerance 11 , it can be concluded that ZmPMP3 genes in the group I have multiple functional features. Increases in leaf RWCs (Fig. 3b) and contents of betaine and total soluble sugars ( Fig. 5) as abiotic stress tolerance-related osmoprotectants 16 along with the decreased leaf wilting index (Fig. 3a) and LWP (Fig. 3c) in transgenic maize lines strongly evidence that ZmPMP3g expression can maintain the cell osmotic potential and water potential under drought. However, on the whole, these parameters' changes were not absolutely proportional to the expression level of ZmPMP3g ( Fig. 1) and even drought tolerance phenotypes (Fig. 2), implying that the ZmPMP3g expression would have the functionally different effects among maize lines differing in genetic backgrounds.
Abiotic stresses usually induce the overproduction of ROS such as O 2 −· and H 2 O 2 , which would cause membrane lipid degradatation and then result in production of MDA [17][18][19] . O 2 −· and H 2 O 2 , can be degraded by SOD and CAT 20    www.nature.com/scientificreports/ Of them, AP2/ERFs, bHLHs, DREB2, and NACs are the downstream genes of ABA-independent signaling pathway, and MYBs, bZIPs and MYCs are the downstream genes of ABA-dependent signaling pathway 28 . Ninecis-epoxycarotenoid dioxygenases (NCED)/viviparous are enzymes responsible for ABA biosynthesis 29,34 and related to ABA-independent signaling pathway by activating NAC proteins 29 . Our results strongly indicate that ZmPMP3g overexpression in transgenic line Y7-1 affects ADIDS pathways by influencing the expression of NCED8, PYL9 (a ABA receptor), viviparous, NAC and AP2/DREB genes (Tables 1 and 2), very similar to actions of Arabidoposis AtRCI2A/B 1 and maize ZmPMP3-1 11 .
It has been found there is a autoregulatory negative feedback path between MYC2 and phytohormone jasmonate to terminate the jasmonate signaling in tomato 35 . The number of DEGs involved in ADIDS pathways obviously tended to decrease in both transgenic Y7-1 ( Table 2) and wildtype Y478 (Supplementary Table S3) under exogenous ABA, indicating that the enhanced ABA levels probably repress expression of the related genes in ADIDS pathways, in turn, the expression of some genes affected by ZmPMP3g overexpression would trigger  www.nature.com/scientificreports/ ABA production. It could be therefore speculated that there exists a autoregulatory feedback path associated with ZmPMP3g overexpression, from ABA production to the gene expression in ADIDS pathways. GAs can relieve from growth restraint by acting degradation of the growth repressor DELLA 36 , of which GA1 and GA3 are major hormones in the cytosol for plant growth 37 . DELLA degradation is also involved in COP1, FKF1/F-box, skp1, CUL1, and F-box proteins 38 . However, accumulating evidence shows that GA and ABA exert an antagonistic effect and need a balance/homeostasis in plants [39][40][41][42] , high GA and low ABA levels under favorable conditions and the reverse ratio under unfavorable conditions [43][44][45] . This is roughly consistent with our findings in this study (Fig. 7d), indicating that ZmPMP3g overexpression can harmonize the ABA-GA1-GA3 (especially GA1) balance, somewhat different from the homeostasis of ABA-GA3 in melon (Cucumis melo) seeds under treatments with exogenous ABA 45 and ABA-GA3-GA4 (especially GA4) during early growth stage of cucumber (C. sativus) treated by melatonin under salt 43 .
In addition, many key regulator and/or receptor genes have been found involving GA-ABA homeostasis, including DELLA, WRKY, ABA-insensitive (ABI)4 (ABI4), ABI5, and GA receptor GID1L2 44,46-50 . Rice OsWRKY24 expression inhibits both GA and ABA signaling 46 . ABI4 expression can enhance the expression of both NCED6 related to ABA biosynthesis and GA2ox7 controlling GA biosynthesis rate-limiting step 47 . Overexpression 48 and repression 49 of ABI5 lead to a hypersensitiy and less sensitivity of Arabidopsis to exogenous ABA, respectively. Rice GID1L2 mutant failes to respond to GA signaling 50 . Taken these reults together with our transcriptome data (Tables 1, 2, 3, 4; Supplementary Tables S1-S3), and changes in ABA and GA levels (Fig. 7d), a working model that the ZmPMP3g overexpression affects growth and drought tolerance of maize was proposed (Fig. 8).

Materials and methods
Maize and ZmPMP3g. Maize lines used were HZ4, C7-2, Y478 and Z58. The maize gene ZmPMP3g was cloned by our laboratory 13 . Pot mix preparation. In brief, the tillage soil from the experimental field of Guangxi University was collected, sun-dried, sieved, and then fully mixed with the local commercial organic fertilizer (8:2/w:w). The prepared mix was loaded in pots (31 cm in diameter and 21 cm in height), 5 kg per pot. The pot mix was saturated with tap water, monitored for moisture by using the Dong mei DT001 soil tester (Shanghai, China) equipped with a thin and long detector following the instructions of the manufacturer, and then started either transplanting plants or sowing maize seeds when the moisture of the pot mix about 1 cm below the surface of the pot mix reached 70%.
ZmPMP3g gene transfer, and Basta screening of transformants. The ZmPMP3g cDNA ranging from start codon to stop codon was cloned into NcoI and BamHI restriction sites downstream of the 3×CaMV35S promoter of plasmid pGSA1252 of Basta resistance gene, generating an expression construct of pGSA1252::ZmPMP3g. The construct was introduced into maize lines thorugh Agrobacterium tumefaciens strain LBA4404 mediated infection of the mature seed embryos. The LBA4404-infected maize embryos were   Table 5. The PCR-amplified DNA was further verified by sequencing. Southern blotting was conducted as the conventional method. In brief, the genomic DNA was digested with HindIII. The DNA was amplified by PCR from the pGSA1252::ZmPMP3g with primers of 35S-F and ZmPMP3g-R (Table 5), and then labelled as DNA probe by using the DIG High Prime DNA Labelling and Detection Starter Kit II (Roche, Sweden).

Analysis of ZmPMP3g expression by qPCR.
The total RNA was isolated by using the TransZol Up Plus RNA Kit (TransGen Biotech, Beijing, China). The first-strand cDNA was synthesized with total RNA by using  [36][37][38]46,47,49,50,[61][62][63][64][65] . Enhanced expression of ZmPMP3g gene would result in three major effects toward the following routes: (1) repressing the expression of some ABI5s, DELLAs, and WRKYs (WRKY2, WRKY6, WRKY32 and WRKY55), and therefore relieving from maize growth restriction caused by elevated ABA; (2) inducing expression of ABI5 and NCED8, promoting ABA production, and consequently endowing maize with drought tolerance; and (3) facilitating expression of GID12 and GA20ox5, and GA1 production, and therefore improving maize growth under drought. In these processes, the cross-talk between the expression of related genes and production of ABA and GA occurred. The red and green boxes indicated an up-regulation/increase and a down-regulation/decrease in gene expression/ABA or GA production, respectively. The dashed lines with arrows denoted that the routes and/or their actions are unknown. The italics indicated genes. ABA, Abscisic acid; ABI5, ABA-insensitive 5; GA, Gibberellin; GA2ox, GA2-oxidase; GA20ox, GA20-oxidase; GID1L2, GA receptor; NCED, Nine-cis-epoxycarotenoid dioxygenase. WRKY, WRKY transcription factor.  www.nature.com/scientificreports/ the PrimeScript™ RT reagent kit containing the gDNA Eraser (TaKaRa, Dalian, China). The first-strand cDNA product was diluted 10 times and then used for qPCR. The qPCR was performed in a 10-μL reaction system containing 2 µL of the 10 times-diluted cDNA product and sequence-specific primers by using the SYBR ® Premix Ex Taq™ II (Tli RNaseH Plus) kit (TaKaRa). The qPCR instrument used was an ABI StepOne Plus™ Real-Time PCR System (Applied Biosystem, Temecula, CA, US). The interner control gene was maize actin gene (GenBamk accession no. XM_008650452.3). The primers used were listed in Table 5 Pot experiments. Mazie seeds were sowed in the pot mix with 70% moisture and grew in the glass greenhouse with conditions indicated above. After emergence, maize seedlings of health and uniform growth were left. During maize growth, the pot mix moisture was monitored daily as mentioned above. If the moisture of the pot mix about 1 cm below the surface of the pot mix was lower than 70%, the pot mix was sprayed with water. When maize seedlings grew to the 5-leaf stage, the pot mix of another group of seedlings was treated for drought by withholding water. Drought degree was determined according to the moisture of the pot mix about 5 cm below the surface of the pot mix, which was defined by reference to the literature 51 and classified into moderate drought of ≤ 50% a mix moisture ≥ 40% and severe drought of < 40% a mix moisture > 30%.
As for the combined treatments of drought/control and ABA, when the pot mix was in the first day of moderate and severe drought, respectively, the maize plants were treated by foilia spraying with 15 mg/L ABA (Sigma, USA) as described by 11 while the seedlings in control treatment were sprayed with an equal volume of tap water.
Total 15 pots were set for each maize line under each treatment. The dates for analyses after treatments were somewhat different according to the specific situation, which were stated in the figure legends and footnotes. However, the tissues were sampled at 10:00 a.m., and/or immediately frozen in liquid nitrogen for analyses. Assay of RWC . The RWC was assayed as described by 52 with modifications. In brief, 1 g of fresh fullyexpanded leaves was cut into small pieces and immersed for 80 min in distilled water. The immersed leaf pieces were collected and then weighed (saturated fresh weight, SFW). After then, the immersed leaf pieces were oven dried to constant weight at 80 °C and then weighed (dry weight, DW). RWC [%] was calculated as a formular of (1 − DW)/(SFW − DW) × 100.

Assay of LWP.
In brief, the surface of fresh fully-expanded leaves was lightly polished by using sandpaper until the leaf mesophyll was exposed. The polished leaves were then immediately detected for LWP by using the WP4-T Dewpoint PotentiaMeteR (METER, US) according to the manufacturer's instructions.
Measurement of total root length. The roots were first scanned by using the Epson Expression 11000XL scanner (Japan) under 1200 dpi and then further analyzed for total root length by using the RhizoVisionExplorer-2.0.3 software of the WinRHIZO root analysis system WinRHIZO software (Regent Instruments Canada Inc. Canada) under built-in default parameters according to the instructions of the manufacturer.
Assay of osmoprotectants. The betaine in the tissues was measured following the high-performance liquid chromatography (HPLC) method 53 but with modifications. Briefly, 1 g of frozen tissues was homogenized in 2 mL of pre-cooled pure methanol by using the scientz-192 tissue grinder (Ningbo, China), The resulting homogenate was diluted to 50 time with Millipore ultra-pure water, concentrated and extracted for 1 h at 75 °C by using a BUCHI R-215 rotary evaporator (Switzerland), cooled on ice, and then filtered with a 0.45-µm filter membrane. The filtrate was then concentrated to a thick paste at 60 °C. The paste was dissolved in 2 mL of ultrapure water, and filtered. The filtrate was then treated for 30 s by ultrasonic degassing. The ultrasonic degassingtreated filtrate was used for betaine analysis by using the Waters e2695 Alliance HPLC system equipped with a Waters 2998 photodiode array detector and the Empower 3.0 software. The internal standard used was analytical pure grade betaine.
The soluble sugars in the tissues were measured following the HPLC-evaporative light-scattering detector method 54 but with modifications. Briefly, 0.1 g of frozen tissues was homogenized in 2 mL of ultra-pure water by using the grinder, and extracted in 80 °C water bath through oscillation for 20 min. The extract was centrifuged at 8000 rpm for 10 min. The resulting supernatant was transferred into a volumetric flask, diluted to 10 mL with ultra-pure water, filtered, and then treated for 30 s by ultrasonic degassing. The ultrasonic degassing-treated filtrate was used for total sugar analysis by using the Waters e2695 Alliance HPLC system equipped with a Waters 2424 evaporative light scattering detector and the Empower 3.0 software. The internal standard used was analytical pure grade d-fructose, d-glucose, and sucrose.
Analysis of MDA. The MDA content in the tissues was determined according to the trichloroacetic acid (TCA)-thiobarbituric acid (TBA) method as described 55  www.nature.com/scientificreports/ samples was fully homogenized in a pre-cooled mortar containing 10 mL of pre-cooled 10% TCA and a small amount of quartz sand on ice. The resulting homogenate was centrifuged at 4 °C and 12,000g for 10 min. The 1 mL of the supernatant resulting from centrifugation was mixed with 1 mL of 0.6% TBA, allowed to react in 100 °C water bath for 15 min, and then quickly cooled to room temperature on ice. was measured following the method described in the literature 57 but with modifications. Frozen tissues (0. 2 g) was fully homogenized in a pre-cooled mortar containing 2 mL of pre-cooled PBS (pH 7.8). The resulting homogenate was centrifuged at 15,000g for 10 min. Four tubes were numbered 0-3 (No. 0-3), of which No. 1-3 tubes were added with 0.5 mL of the resulting homogenate respectively, and No.0 tube was added with 0.5 mL of distilled water. Then, 0.5 mL of 50 mM PBS and 1 mL of 1 mM hydroxylamine hydrochloride were added to each tube. The tubes were placed in 100 ºC water bath for 1 h, and then quickly cooled to room temperature on ice. Each tube was then added with 1 mL of 17 mM p-aminobenzene sulfonic acid and 1 mL of 7 mM α-naphthylamine, and placed in 25 °C water bath for 25 min. The OD value of the resulting reaction mixture in each tube was measured for estimation of O 2 −· content at 530 nm by using the NanoDrop 2000c spectrophotometer. The content of O 2 −· in the solution was calculated against the NO 2− concentration in a calibration curve plotted with sodium nitrite satandads and then converted into the H 2 O 2 content in the tissues as μM g −1 FW.

Analysis of activities of SOD and CAT enzymes.
For each sample, 0.2 g of the tissues was homogenized in 5 mL of 50 mM pre-cooled PBS (pH 7.8), transfered to a tube and then centrifuged for 20 min at 12,000g at 4 °C. The resulting supernatant was used as the crude proteinase extract for analyses of SOD and CAT activity.
The SOD activity was analyzed as the method described in the literature 58 but with modifications. In brief, the reaction mixture was prepared with PBS (pH 7.8), which was composed of 1.3 μM riboflavin, 13 μM methionine, 63 μM NBT, 0.05 M sodium carbonate (pH 10.2). The 3 mL of reaction mixture were fully mixed with 30 μL of the extract. The mixture was then illuminated for 20 min under a light of 4000 lx in glass test tube. The glass test tubes that were added with both 3 mL reaction mixture and 30 μL PBS served as control. After illumination, the OD value of the resulting reaction mixture was measured for estimation of SOD activity at 530 nm in dark by using the NanoDrop 2000c spectrophotometer.
The CAT activity was analyzed as the method described in the literature 59 but with modifications. In brief, the reaction mixture was composed of 457.2 mL of 0.15 mM PBS (pH 7.0) and 0.3092 mL of 30% H 2 O 2 . The 3 mL of reaction mixture were fully mixed with 30 μL of the extract, immediately read for OD values once at 240 nm every 30 s for total 5 min. The decrease of OD 240 value by 0.01 per minute was defined as one enzyme activity unit (U).
Analysis of endogenous ABA and GA. The ABA and GA content in the tissues was detected by Met-Ware (http:// www. metwa re. cn/) based on the AB Sciex QTRAP 6500 LC-MS/MS platform following the methods decribed in the literature 60 and conducted as the in-house protocols provided by the Genedenovo Biotechnology Co., Ltd (Guangzhou, China). In brief, the frozen tissue samples were ground to powder by using the MM400 ball mill (Retsch, Germany) at 30 Hz for 1 min. The 50 mg of the powder were fully mixed with an appropriate amount of internal standard and 1 mL of methanol/water/formic acid (15:4:1, v/v/v). The resulting extraction solution was further concentrated by using the CentriVap vacuum concentrator (LABCONCO, US). The concentrate was re-dissolved with 100 μ L 80% methanol/water solution, and filtered with a 0.22-μm filter membrane for LC-MS/MS analysis. The LC-MS/MS analysis was conducted as the following conditions: 550 °C electrospray ionization, 5,500 V MS voltage in positive ion mode, − 4,500 V MS voltage in negative ion mode, and 35 psi curtain gas. In Q-Trap 6500+, each ion pair was scanned and detected according to the optimized de-clustering potential and collision energy.
Transcriptome sequencing. Transcriptome sequencing was based on the sequencing platform of the Novogene (Beijing, China). In brief, RNA was isolated from the second fresh leaves down from tops of plants. RNA degradation and contamination were monitored on 1% agarose gels. RNA purity was checked by using the NanoPhotometer ® spectrophotometer (IMPLEN, CA, USA). RNA integrity was assessed by using a RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system. A total of 1 µg RNA for each sample was used for construction of sequencing libraries. The libraries were generated by using a NEBNext ® UltraTM RNA Library Prep Kit for Illumina ® (NEB, USA) and sequenced by using the Illumina Novaseq platform. The transcriptome for each maize line under each treatment was repeated with the second fully-expanded leaves from 3 individual plants.

Statement of plants involved in this research.
The wild type maize materials used in this study were from the Institute of Crop Sciences, CAAS, China, which comply with relevant institutional, national, and international guidelines and legislation in China.

Data availability
All data are available in the text and supplementary data without undue reservation. The transcriptome datasets generated and/or analyzed during the current study are available in the NCBI's GEO repository under accession number GSE220124 (https:// www. ncbi. nlm. nih. gov/ geo/ info/ linki ng. html).